C6 loaded-NPs were fabricated with the following surface
modifications: 1) unmodified (NP-C6-Unmod), 2) avidin-only modified
(NP-C6-AV), 3) AP-modified (NP-C6-AP), 4) 2.5 kDa chitosan-modified
(NP-C6-CH2.5), and 5) 20 kDa chitosan-modified (NP-C6-CH20). Peptides were
synthesized by the W.M. Keck Facility at Yale University, and contained a
Ser-Gly dipeptide spacer between the N-terminal amino acid and biotin.
Prior to NP synthesis, avidin-palmitate was conjugated to the surface
of PLGA-NPs (50:50 carboxy-terminated, inherent viscosity range
0.55–0.75 dL/g, LACTEL®) as previously described
(14 (link)). Briefly, the
avidin-palmitate was prepared by reacting 10 mg of avidin with 14-fold molar
excess of palmitic acid-N-hydroxysuccinimide ester in 2% sodium
deoxycholate in phosphate buffered saline (PBS) (37°C, 12 hr),
followed by overnight dialysis.
C6 NPs were synthesized using a single oil-in-water emulsion
technique (14 (link), 23 (link)). One hundred mg PLGA were dissolved overnight in 1
mL methylene chloride (DCM) (oil). C6 (Acros Organics, Geel, Belgium) was
added to the DCM polymer solution (15 µg C6/mg polymer), vortexed
and sonicated. For unmodified NPs, the PLGA-C6 solution was added dropwise
to 2.5% polyvinyl alcohol (PVA) alone (aqueous), whereas for
modified NPs, the PLGA-C6 solution was added to 2.5% PVA containing
1 mg/mL avidin palmitate. Both solutions were vortexed and sonicated. After
the sonication/emulsion step, the emulsified solution was added to a
0.3% PVA mixture while stirring to evaporate the DCM. After 3 hours,
this solution (DCM evaporated, leaving NPs in PVA) was added to centrifuge
tubes and centrifuged/washed three times with DI water to remove the PVA.
After 3 washes/centrifuges the NPs were suspended in 5–10 mL of DI
water, frozen at −80°C, and subsequently lyophilized to
produce dry NPs.
After NPs were hardened during solvent evaporation, those with
surface modification were reacted with ten times molar excess biotin-CH2.5,
CH20 or AP to avidin, in PBS for 30 min. Unmodified NPs were incubated in
PBS for 30 min without ligand. After the reaction, NPs were washed 2 times
in deionized water to remove residual solvent, centrifuged at 4° C,
lyophilized, and stored at −20°C until use.
modifications: 1) unmodified (NP-C6-Unmod), 2) avidin-only modified
(NP-C6-AV), 3) AP-modified (NP-C6-AP), 4) 2.5 kDa chitosan-modified
(NP-C6-CH2.5), and 5) 20 kDa chitosan-modified (NP-C6-CH20). Peptides were
synthesized by the W.M. Keck Facility at Yale University, and contained a
Ser-Gly dipeptide spacer between the N-terminal amino acid and biotin.
Prior to NP synthesis, avidin-palmitate was conjugated to the surface
of PLGA-NPs (50:50 carboxy-terminated, inherent viscosity range
0.55–0.75 dL/g, LACTEL®) as previously described
(14 (link)). Briefly, the
avidin-palmitate was prepared by reacting 10 mg of avidin with 14-fold molar
excess of palmitic acid-N-hydroxysuccinimide ester in 2% sodium
deoxycholate in phosphate buffered saline (PBS) (37°C, 12 hr),
followed by overnight dialysis.
C6 NPs were synthesized using a single oil-in-water emulsion
technique (14 (link), 23 (link)). One hundred mg PLGA were dissolved overnight in 1
mL methylene chloride (DCM) (oil). C6 (Acros Organics, Geel, Belgium) was
added to the DCM polymer solution (15 µg C6/mg polymer), vortexed
and sonicated. For unmodified NPs, the PLGA-C6 solution was added dropwise
to 2.5% polyvinyl alcohol (PVA) alone (aqueous), whereas for
modified NPs, the PLGA-C6 solution was added to 2.5% PVA containing
1 mg/mL avidin palmitate. Both solutions were vortexed and sonicated. After
the sonication/emulsion step, the emulsified solution was added to a
0.3% PVA mixture while stirring to evaporate the DCM. After 3 hours,
this solution (DCM evaporated, leaving NPs in PVA) was added to centrifuge
tubes and centrifuged/washed three times with DI water to remove the PVA.
After 3 washes/centrifuges the NPs were suspended in 5–10 mL of DI
water, frozen at −80°C, and subsequently lyophilized to
produce dry NPs.
After NPs were hardened during solvent evaporation, those with
surface modification were reacted with ten times molar excess biotin-CH2.5,
CH20 or AP to avidin, in PBS for 30 min. Unmodified NPs were incubated in
PBS for 30 min without ligand. After the reaction, NPs were washed 2 times
in deionized water to remove residual solvent, centrifuged at 4° C,
lyophilized, and stored at −20°C until use.
